Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has become one of the most popular methods for the rapid, cost-effective and accurate classification and characterization of cultivable microorganisms. Due to its simple sample preparation and short measurement time, MALDI-TOF MS is an excellent choice for the high-throughput study of microbial isolates from rhizospheres or plants grown under diverse environmental conditions. While clinical isolates have a higher identification rate than environmental isolates due to the focus of commercial mass spectral libraries on the former, no identification is necessary in the dereplication step of large environmental studies. The grouping of large sets of isolates according to their intact protein profiles can be performed without knowledge of their taxonomy. Thus, this method is easily applicable to environmental samples containing microorganisms from yet undescribed phylogenetic origins. The main strategies applied to achieve effective dereplication are, first, expanding existing mass spectral libraries and, second, using an additional statistical analysis step to group measured mass spectra and identify unique isolates. In this review, these aspects are addressed. It closes with a prospective view on how MALDI-TOF MS-based microbial characterisation can accelerate the exploitation of plant-associated microbiota.
Trang 1Rapid dereplication of microbial isolates using matrix-assisted laser
desorption ionization time-of-flight mass spectrometry: A mini-review
Doreen Huscheka, Katja Witzelb,⇑
a German Rheumatism Research Centre – A Leibniz Institute, Charitéplatz 1, 10117 Berlin, Germany
b
Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany
h i g h l i g h t s
MALDI-TOF MS is applicable as
high-resolution and high-throughput tool
The classification and
characterization of cultivable
microorganisms is targeted
Advantageous are its simple sample
preparation and short measurement
time
It accelerates the dereplication of
isolates from large-scale screening
campaigns
Applications for studying microbial
diversity and future trends are
discussed
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:
Received 16 December 2018
Revised 20 March 2019
Accepted 21 March 2019
Available online 2 April 2019
Keywords:
Dereplication
MALDI-TOF MS
Environmental isolates
Data analysis
Expansion of mass spectral databases
a b s t r a c t
Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) has become one of the most popular methods for the rapid, cost-effective and accurate classification and characterization of cultivable microorganisms Due to its simple sample preparation and short measure-ment time, MALDI-TOF MS is an excellent choice for the high-throughput study of microbial isolates from rhizospheres or plants grown under diverse environmental conditions While clinical isolates have a higher identification rate than environmental isolates due to the focus of commercial mass spectral libraries on the former, no identification is necessary in the dereplication step of large environmental studies The grouping of large sets of isolates according to their intact protein profiles can be performed without knowledge of their taxonomy Thus, this method is easily applicable to environmental samples containing microorganisms from yet undescribed phylogenetic origins The main strategies applied to achieve effective dereplication are, first, expanding existing mass spectral libraries and, second, using
an additional statistical analysis step to group measured mass spectra and identify unique isolates In this review, these aspects are addressed It closes with a prospective view on how MALDI-TOF MS-based microbial characterisation can accelerate the exploitation of plant-associated microbiota
Ó 2019 The Authors Published by Elsevier B.V on behalf of Cairo University This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Introduction to MALDI-TOF MS-based microbial characterisation
Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is an advanced tool for the fast and high-resolution characterization of microorganisms [1] The
https://doi.org/10.1016/j.jare.2019.03.007
2090-1232/Ó 2019 The Authors Published by Elsevier B.V on behalf of Cairo University.
Peer review under responsibility of Cairo University.
⇑ Corresponding author.
E-mail address: witzel@igzev.de (K Witzel).
Contents lists available atScienceDirect
Journal of Advanced Research
j o u r n a l h o m e p a g e : w w w e l s e v i e r c o m / l o c a t e / j a r e
Trang 2method is based on measurement of the molecular mass of ions
generated from the most abundant proteins of a sample culture
and uses the mass spectral information as a fingerprint for a
partic-ular organism (exemplified inFig 1) A typical workflow
contain-ing MALDI-TOF MS starts with the isolation of microorganisms
from a chosen sample and their cultivation on nutrient medium
to obtain a pure axenic culture[2–4] Microbial cultures in their
exponential phase are grown under standardized conditions and
then subjected to sample processing in two different ways: a direct
method or a solvent extraction method The first is a fast technique
where a smear of microbial cells is applied directly to the MALDI
target plate This approach usually leads to low-quality spectra
due to overloading or the presence of compounds disturbing the
ionization process, but can be recommended for routine
assess-ments For acquisition of high-quality spectra, cell walls are lysed
in a suitable way, and proteins are extracted with (usually) formic
acid using the solvent extraction method (Fig 2) The samples
spotted onto the MALDI target plate are then overlaid with matrix,
and spectra are acquired from intact proteins in the range of m/z
2000 – 20,000[5] These spectra are then matched to a reference
library to determine the identity of the microorganism There are
several vendors on the market providing instrumental and
soft-ware solutions as well as commercial spectral libraries for
MALDI-TOF MS-based biotyping [6] As an example, the Bruker
MALDI Biotyper library contains spectra of 7014 bacterial and
1300 filamentous fungi isolates (as on February 1st, 2019)
The popularity of MALDI-TOF MS for microbial biotyping is
based on its speed, simplicity and cost efficiency Due to these
advances, MALDI-TOF MS diagnosis has been successfully adapted
to clinical microbiology in the past 20 years to accelerate patient
diagnosis and therapy[7]and plays a vital role in the
characterisa-tion of human gut microbiota [8] Constant enhancements in instrumental platforms, sample processing methods and extent
of spectral libraries accelerated the establishment of MS-based diagnosis in clinical laboratories and readers are referred to com-prehensive reviews regarding its clinical application[1,9] In some cases, acquired MALDI-TOF spectra are used to create dendrograms and establish pseudo-phylogenetic groupings based on the similar-ity of mass spectra However, because MS spectra, having a limited number of peaks, lack the evolutionary relatedness of small-subunit rRNA sequences or other genomic information, a determi-nation of relatedness of unknown isolates is difficult but can be facilitated by combined analysis of additional biomarkers[10,11] The method is applicable for a wide range of microbial isolates, including those of bacteria[12–14], fungi[15]and archaea[16], and extends to many other cultivable organisms, such as microal-gae[17], protozoa[18]or viruses[19] Although MALDI-TOF MS is successfully applied in the identification of clinical isolates[2,20], characterization of isolates from plant-associated samples is ham-pered by a lack of reference spectra in available databases Never-theless, MALDI-TOF MS as a powerful tool for the rapid grouping of bacterial isolates, i.e., dereplication, in large-scale screening cam-paigns In this review, the applicability of this method as a high-resolution tool for studying microbial diversity is discussed
MS-based exploration of plant-associated microbial communities
With an increased understanding of the diversity of plant-bacterial associations, future biotechnological applications for stable crop production, conservation of biodiversity and
sustain-Fig 1 Exemplary MALDI-TOF MS profiles of three plant-associated bacterial species showing the heterogeneity of protein profiles The sequence and intensity of mass peaks,
Trang 3able agro-ecosystems are foreseeable Hence, there is a high
demand for high-throughput methods for the classification and
characterization of cultivable microorganisms isolated from soils,
rhizospheres or plants grown under diverse environmental
condi-tions There is a growing awareness of the complex interplay
between plants, soil and their microbial communities, and current
research efforts aim at understanding how microbiota present in
rhizospheres and endospheres of crops account for plant health
and productivity[21–23] Up to the present, microbial
communi-ties were described often by shotgun sequencing approaches,
which left their functionalities and activities aside More recently,
in order to close this gap, microbes have been isolated from their
respective environments in extensive culture experiments and
assessed for their physiological properties [24] Novel nutrient
media are developed to allow the isolation of niche
microorgan-isms [25,26] A long-term goal is to manage and engineer soil
microorganisms by agricultural practice, to select proper plant
genotypes or to apply microbial inoculants with a distinct function,
such as biocontrol, growth promotion or abiotic stress alleviation
[27]
A common strategy in studying cultivable microorganisms is
plating the chosen disrupted tissue or sample on culture medium
and assessing the growth of developing colonies Then,
morpholog-ically different colonies are selected for further analysis such as
16S rRNA sequencing or biochemical testing [28,29–32] This
approach usually leads to a bias in assessing the diversity of a
habi-tat since morphological similar species, that may have different
metabolic capabilities, are excluded from downstream
investiga-tions Another way of conducting such ecological experiments is
to process all isolated microbes for nucleotide analysis without
preselection, which results in a considerable sample load and high expense [33–35] The application of MALDI-TOF MS for fast and inexpensive dereplication of recurrent isolated microorganisms would be of particular advantage in large microbial community studies since the grouping of large sets of isolates according to their intact protein profiles can be performed without knowledge
on their taxonomic identification
Previous studies demonstrated the applicability of MALDI-TOF
MS for high-throughput dereplication and its applicability for unbiased studies of the cultivable microbial community[36–39] The discrimination power of MALDI-TOF MS by combining MS data from both intact proteins and specialized metabolites was recently demonstrated and allowed the characterisation of isolates based
on their identity and potential environmental function [10] In the following, an overview of the bioinformatic background of the dereplication principle is provided
Bioinformatic means for dereplication of microbial isolates Identification of plant-associated isolates is less successful as compared to clinical microorganisms due to an underrepresenta-tion of environmental reference strains in commercial mass spec-tral databases [40,41] Two main bioinformatic strategies are commonly used to improve dereplication when using whole-cell
or simple acidic protein extracts for MALDI-TOF MS The first strat-egy is to expand the commercial databases by including additional plant-associated reference strains This approach has the advan-tage of still profiting from the simplicity of sample preparation and rapidness in measurement and identification of the MALDI biotyping as no additional statistical analysis is required, but
Fig 2 The effect of sample processing on quality of mass spectra Application of a protein extraction method (upper panel) results in higher number of detected peaks and better signal-to-noise ratios as compared to the direct transfer method (lower panel) The direct transfer of bacterial cells to the MALDI target gives higher background signals, but the quality of spectra might be sufficient for routine analysis.
Trang 4achieves better identification rates[16,42] Most platforms have
options for researchers to customize the mass spectral libraries
with user-selected reference strains and provide training or
proto-cols to create a personalized database Suitable reference strains
can be (1) cultivated environmental samples that have not been
identified, (2) purchased, cultivated and measured known
refer-ence strains and (3) strains whose mass spectra have been received
from other institutes While some attempts were made to create
open-access repositories, they are still very limited in scope[15]
Often, commercial libraries allow only genus-level identification
for plant-associated samples The accuracy of identification can
generally be improved by including in-house reference spectra,
as they are measured with the same techniques, technicians and
machines In the context of expanding a spectral library, it is
cru-cial to realize that confident species or strain identification can
be achieved only when several reference strains of one species
are available in the database Usually, only one strain per species,
with the exception of the most common clinical bacteria, is present
in the commercial databases To improve identification, it is
advised by commercial library vendors that three to six strains
for one species that take into account biological variations should
be included for common environmental microorganisms However,
these library expansions need continuous work, and their
mainte-nance can be time consuming
In large cohort microbial studies, identifying the number of
unique species or strains can also be achieved without identifying
a microorganism For the dereplication step, grouping isolates from
the same taxon rapidly to determine and reduce the number of
iso-lates for further analysis is sufficient[36] Therefore, the second
strategy involves using statistical analysis to group mass spectra
A first step can be to use available opportunities for visualisation
provided by commercial software to create for instance
dendro-grams or composite correlation matrices[43]to determine similar
isolates from all the isolates of one study (Fig 3) These methods
are performed by clustering the obtained peak mass lists or the
whole mass spectra of different isolates However, it can be
diffi-cult to visually decide whether individual clusters in a dendrogram
represent isolates from the same species or what level of correla-tion between mass spectra represents isolates from the same spe-cies Using additional statistical analysis steps or software with further options can therefore improve the approach in creating a nonredundant set of isolates Several studies have successfully implemented MALDI-TOF MS-based biotyping to classify mass spectra for dereplication[36–39,44] Generally, highly similar clus-ters were used to identify identical isolates and select representa-tives of these clusters for further validation, e.g., via partial 16S rRNA gene sequence analysis, ITS region sequencing or repetitive element-based PCR The evaluation of appropriate cut-off values for cluster delineation was based on threshold values established
by the additional validation steps and/or a minimum number of mass peaks shared between isolates It was shown that a similarity-based MALDI-TOF MS approach can be used for derepli-cation without additional, costly DNA-based methods[38] New approaches also include machine learning algorithms such as Ran-dom Forest models to automate the identification of isolates in environmental studies[45,46]
Current issues and implications for using MS-based biotyping The quality of mass spectra is important for successful MALDI-TOF MS-based analysis of microorganisms For example, that iden-tification can be improved in various ways other than adding miss-ing or rare species to the database or optimizmiss-ing pre-analytical settings (e.g., sample preparation, growth conditions, and matrix use[5,47–49]) A quality check of the acquired mass spectra and
of the pre-set parameters for automated data acquisition is essen-tial, especially when using automated measuring tools Including low-quality spectra can lead to false positive identification or no identification Common problems include suppressed peaks, low peak intensities, and matrix background signals Quality checks need to be included, especially when reference mass spectra for libraries are created It was suggested that a good spectrum should have a minimum of 70 peaks for bacteria and 30–40 peaks for fungi and an average peak intensity of 103arbitrary units or higher[41]
Fig 3 Approaches for visualizing the relationship between mass spectra derived from microbial samples Protein extracts from 36 bacterial isolates, originated from parsley phyllosphere, were analysed by MALDI-TOF MS Recurrent isolated microorganisms can be identified by cluster analysis of protein patterns, where the height of each node is proportional to the dissimilarity value (A) In a composite correlation index matrix, the degrees of mass spectrum correlation are indicated by colour coding (dark red = closely related, dark blue = not closely related) and scoring from 0 to 1, where 1 is an exact match (B) Experimental sample set was kindly provided by Dr Silke Ruppel, Leibniz Institute of Vegetable and Ornamental Crops, Germany.
Trang 5To reduce variations caused by technicians and instruments,
sam-ples should be spotted in triplicate, and each spot should be
anal-ysed at least three times In a comparison of manual and automatic
mass spectrum measurements, it was found that while automatic
measurement tended to increase the base peak resolution, other
measures of spectrum quality such as signal-to-noise ratio, data
richness and reproducibility were reduced [50] Looking at the
same issue of the low reproducibility of automated spectrum
acquisition, another study reported optimized user threshold
val-ues of several parameters (peak selection mass range,
signal-to-noise ratio, threshold peak intensity, threshold minimum
resolu-tion, and number of shots summed) and improved reproducibility
[51]
A further aspect to consider is that MALDI-TOF MS requires a
monoculture to perform identification However, morphologically
similar samples do not need to contain the same species In
envi-ronmental dereplication studies, a re-cultivating step from a single
colony to control for a pure culture may not be performed
There-fore, some unidentified spectra may be bacterial mixtures that can
be hard to identify with algorithms developed to identify single
microorganisms The resulting mass spectra often have signals
with suppressed intensity However, the main abundant peaks of
all the species should be present but at much lower intensity than
peaks from samples from pure cultures Further statistical analysis
could be used to identify these spectra as well The large variation
in peaks and intensities occurring with mixtures is an issue Only a
few studies have attempted to address this challenge by applying
different biomarker identification strategies [52–54] However,
this methodology is yet to be standardized and mixtures of
microorganisms are more frequently analysed via tandem MS
strategies[55]
Conclusions and future perspectives
MALDI-TOF MS is the method of choice for the grouping of
plant-associated microbial isolates due to its fast, simple and
cost-effective measurement of a large number of samples Recent
developments in automation of colony picking and deposition on
the MALDI target as well as matrix deposition should further
decrease consumable costs and preparation time[56] An increased
use of MALDI-TOF MS in large-scale screening campaigns to collect
microorganisms from rhizospheres or plants is going to lead to the
detection of novel species that could bear a potential use to
sus-tainably increase crop production [57] Future improvements in
dereplication, either by expanding commercial mass spectral
libraries and/or by implementing an additional data analysis step
to group mass spectra, are expected to further exploit the capacity
of MALDI-TOF MS in microbial studies
Conflict of interest
The authors have declared no conflict of interest
Compliance with Ethics Requirements
This article does not contain any studies with human or animal
subjects
Acknowledgements
We thank Dr Silke Ruppel for providing the experimental
sam-ple set depicted in Fig 3 Funding by the Federal Ministry of
Research and Education, Germany (SproutMO, FKZ 01DH14009)
is gratefully acknowledged
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[57] Spitaels F, Wieme AD, Vandamme P MALDI-TOF MS as a novel tool for dereplication and characterization of microbiota in bacterial diversity studies In: Demirev P, Sandrin TR, editors Applications of mass spectrometry in microbiology: From strain characterization to rapid screening for antibiotic resistance Cham: Springer International Publishing; 2016 p 235–56 Doreen Huschek is a statistician at the DRFZ (German Rheumatism Research Center Berlin), Germany She studied demography at the University of Rostock and the Max Planck Institute for Demographic Research In 2011, she received her PhD degree from the Free University Amsterdam Her current research interests include epidemiology, biostatistics and proteomic analysis.
Katja Witzel is working as a scientist on plant-pathogen interactions at the IGZ Leibniz Institute of Vegetable and Ornamental Crops, Germany She holds a PhD in Plant Physiology from the University of Halle/Wittenberg, Germany Her research focuses on characterizing regu-latory networks in crop biotic and abiotic stress defence
at the proteome level The study of plant-microbe interactions, particularly in the rhizosphere, has become another major topic.